2.1. Gene Therapy
Recent progress in the areas of molecular biology and genetic engineering have led to the isolation and characterization of many genes associated with genetic diseases. This in turn has led to the development of the concept of gene therapy i.e., the replacement or supplementation of defective genetic information by transfer of normal functional genes, as a potential method for treating genetic disorders.
Currently available methods for transfer of genes into cells include chemical techniques such as direct calcium phosphate coprecipitation of DNAs into cells; mechanical techniques such as microinjection of cells with genetic materials; and membrane fusion-mediated transfer of genes via liposomes. The principle disadvantage associated with each of these techniques is that they are not practical for in vivo gene therapy applications, and the transfer of genetic material is often non-stable, and successful transfer is unpredictable. In an attempt to circumvent this problem, recent approaches for gene therapy have involved gene transfer using recombinant viral vectors, exploiting RNA and DNA tumor viruses. Many of these viruses are pathogenic and capable of causing disease. It has been proposed that these viruses could be genetically manipulated to deprive them of deleterious characteristics while maintaining their usefulness for possible introduction of stable, inheritable and functional genetic information by infection of cells.
The retroviruses represent one class of viruses that have been extensively studied for use in gene therapy (Miller, A.D., 1990, Human Gene Ther. 1:5-14). Unfortunately, there are a number of disadvantages associated with retroviral use, including the random integration of retroviruses into the host genome which may lead to insertional mutagenesis, or the inadvertent activation of protooncogene expression due to the promoter activity associated with retroviral LTRs (long terminal repeats). The Adeno-associated viruses have also been studied as an alternative system for delivery of stable genetic information into the cell. These viruses have the desirable feature of potentially integrating in specific regions of the host genome. However, the usefulness of both retroviral and AAV vectors is limited by their inability to accept heterologous DNA fragments greater than 3-5 Kb, the inability to produce larger quantities of viral stocks, and in the case of retroviruses, instability.
2.2. Adenovirus Based Vectors
Adenovirus (Ad) is a large, nonenveloped virus consisting of a dense protein capsid and a large linear (36 kb) double strand DNA genome. Adenovirus infects a variety of both dividing and non-dividing cells, gaining entry by receptor-mediated uptake into endosomes followed by internalization. After uncoating, the Ad genome expresses a large number of different gene products that are involved in viral replication, modification of host cell metabolism and packaging of progeny viral particles. Three Ad gene products are essential for replication of viral genomes: the terminal binding protein which primes DNA replication, the viral DNA polymerase, and the DNA binding protein (reviewed in Tamanoi and Stillman, 1983, Immunol. 109:75-87). In addition, processing of the terminal binding protein by the Ad 23kDa L3 protease is required to permit subsequent rounds of reinfection (Stillman et al., 1981, Cell, 23:497-508) as well as to process Ad structural proteins, permitting completion of self-assembly of capsids (Bhatti and Weber, 1979, Virology, 96:478-485).
Packaging of nascent Ad particles takes place in the nucleus, requiring both cis-acting DNA elements and trans-acting viral factors, the latter generally construed to be a number of viral structural polypeptides. Packaging of adenoviral DNA sequences into Ad capsids requires the viral genomes to possess functional Ad encapsidation signals, which are located in the left and right termini of the linear viral genome (Hearing et al., 1987, J. Virol. 61:2555-2558). Additionally, the packaging sequence must reside near the ends of the viral genome to function (Hearing et al., 1987, J. Virol. 61:2555-2558; Grable and Hearing, 1992, J. Virol., 66:723-731). The E1A enhancer, the viral replication origin, and the encapsidation signal compose the duplicated inverted terminal repeat (ITR) sequences located at the two ends of Ad genomic DNA. The replication origin is loosely defined by a series of conserved nucleotide sequences in the ITR which must be positioned close to the end of the genome to act as a replication-priming element (reviewed in Challberg and Kelly, 1989, Biochem, 58:671-717; Tamanoi and Stillman, 1983, Immunol. 109:75-87). As shown by several groups, the ITRs are sufficient to confer replication to a heterologous DNA in the presence of complementing Ad functions. Ad “mini-chromosomes” consisting of the terminal ITRs flanking short linear DNA fragments (in some cases non-viral DNAs) were found to replicated in vivo at low levels in the presence of infecting wild-type Ad, or in vitro at low levels in extracts prepared from infected cells (e.g., Hay et al., 1984, J. Mol. Biol. 175:493-510; Tamanoi and Stillman, 1983, Immunol. 109:75-87). Evidence for trans-packaging of mini-chromosomes was not reported in these or any later studies concerned with mechanisms of Ad DNA replication, and it is unlikely that packaging occured for several reasons. First, the replicated molecules were quite small, and they were not expressed at levels high enough to compete for packaging. Second, no selection for trans-packaging was employed, making it inconceivable that the heterologously replicated molecules could compete for packaging against wild-type Ad genomes.
The expression of foreign genes in “replication-defective” Ad viruses (deleted of region E1) has been exploited for a number of years in many labs, and a variety of published reports describe several different approaches often used in constructing these vectors (Vernon et al., 1991, J. Gen. Virol., 72:1243-1251; Wilkinson and Akrigg, 1992, Nuc. Acids Res., 20:2233-2239; Eloit et al., 1990, J. Gen. Virol., 71:2425-2431; Johnson, 1991; Prevec et al., 1990, J. Infect. Dis., 161:27-30; Haj-Ahmad and Graham, 1986, J. Virol., 57:267-274; Lucito and Schneider, 1992, J. Virol., 66:983-991; reviewed in Graham and Prevec, 1992, Butterworth-Heinemann, 363-393). In general, replication-defective viruses are produced by replacing part or all of essential region E1 with a heterologous gene of interest, either by direct ligation to viral genomes in vitro, or by homologous recombination within cells in vivo (procedures reviewed in Berkner, 1992, Curr. Topics Micro. Immunol., 158:39-66). These procedures all produce Ad vectors that replicate in complementing cell lines such as 293 cells which provide the E1 gene products in trans. Replication competent Ad vectors have also been described that have the heterologous gene of interest inserted in place of non-essential region E3 (e.g., Haj-Ahmad and Graham, 1986, J. Virol. 57:267-274), or between the right ITR and region E4 (Saito et al., 1985, J. Virol., 54:711-719). In both replication defective viruses and replication competent viruses, the heterologous gene of interest is incorporated into viral particles by packaging of the recombinant Ad genome. To demonstrate the feasibility of correcting defects in α-1 AT deficiency or cystic fibrosis, replication-defective Ad vectors expressing the α-1 antitrypsin gene and the CFTR gene, respectively, were used to deliver foreign genes to the lungs of cotton rats by injection of viruses (Rosenfeld et al., 1991, Science, 252:431-434; Rosenfeld et al., 1992, Cell, 252:431-434; Zabner et al., 1993, Cell 75:207-216). In addition, a replication-defective Ad vector expressing β-galactosidase, in place of the region E1, was directly injected into mouse brain and found to nonproductively infect glial and neuronal cells (LaSalle et al., 1993, Science, 259:988-990).
A number of potential drawbacks can be attributed to the use of currently available adenovirus systems, such as the strict controls on the size of the genome that can be packaged. Adenovirus genomes cannot exceed 103-104% the normal 36 kb length (i.e. −2 kb extra; Ghosh-Choudhury et al., 1987, EMBO J. 6:1733-1739). Therefore, recombinant viruses deleted of region E1 and/or E3 can only accommodate foreign DNA inserts of up to 6-7 kb (reviewed in Berkner 1992, supra). This precludes the use of Ad vectors for introduction of large DNA fragments such as genomic DNAs and complex regulatory units generally required for sophisticated tissue expression.
In addition, conventional replication-defective Ad recombinants which have the E1 region deleted, have been used with the belief that deletion of region E1 (either partially or entirely) will prevent expression of other Ad genes. Although region E1 is required to activate expression of the Ad genome, there is a body of evidence indicating that at high multiplicities of infection or in certain cell types, viral replication and cytopathic effects (CPE) are occasionally observed even in the absence of E1A gene expression (e.g., Eloit et al., 1990, J. Gen. Virol, 71:2425-2431; Postlethwaite, 1973, Scott. Med. J., 18:131).
Another potential drawback is that early Ad genes other than E1A have been shown to cause pathology in the lungs of cotton rats, which have served as an animal model for human infection. Infection with a virus that expresses Ad early regions but does not replicate causes significant cytopathology, although not as severe as wild-type replicating viruses (Ginsberg et al., 1990, Proc. Natl. Acad. Sci. USA, 87:6191-6195). Finally, with the currently available Ad systems the potential exists for creation of an “escape” wild-type virus resulting from recombination between the recombinant Ad vector and natural infection by helper viral DNA sequences. Recombination during natural infection between the vector and endogenous Ads can result in restoration of autonomous replication to the vector and creation of unanticipated viral variants, a real and alarming possibility inherent in current gene therapy applications.
Trans-packaging systems specific for Ad-recombinants might solve a number of the drawbacks associated with the use of adenoviral systems. However, trans-packaging systems specific for Ad-based genomes have not been described before. Although bacteriophage packaging systems have been in wide use for years (e.g., Feiss and Becker, 1983, Lambda II, Cold Spring Harbor Press, 305-330), unlike bacterial viruses, the ability to trans-package Ad DNA genomes appears to be more complicated and precisely controlled, and approaches used successfully for bacteriophage systems have not worked for Ad (Kosturko and Vanech, 1986, Virus Res., 6:123-132).
Several studies have been published attempting to produce in vitro (cell-free) extracts for trans-packaging of heterologous DNAs into empty Ad virus capsids, akin to those described for bacteriophage systems (Tibbetts and Giam, 1979, J. Virol. 32:95-105; Kosturko and Vanech, 1986, Virus Res., 6:123-132). However, specific encapsidation of heterologous DNAs into Ad capsids has not been achieved.